Kath James E, Chang Seungwoo, Scotland Michelle K, Wilbertz Johannes H, Jergic Slobodan, Dixon Nicholas E, Sutton Mark D, Loparo Joseph J
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Department of Biochemistry, University at Buffalo, State University of New York, Buffalo, NY 14214, USA Witebsky Center for Microbial Pathogenesis and Immunology, University at Buffalo, State University of New York, Buffalo, NY 14214, USA.
Nucleic Acids Res. 2016 Feb 29;44(4):1681-90. doi: 10.1093/nar/gkv1375. Epub 2015 Dec 10.
Escherichia coli has three DNA polymerases implicated in the bypass of DNA damage, a process called translesion synthesis (TLS) that alleviates replication stalling. Although these polymerases are specialized for different DNA lesions, it is unclear if they interact differently with the replication machinery. Of the three, DNA polymerase (Pol) II remains the most enigmatic. Here we report a stable ternary complex of Pol II, the replicative polymerase Pol III core complex and the dimeric processivity clamp, β. Single-molecule experiments reveal that the interactions of Pol II and Pol III with β allow for rapid exchange during DNA synthesis. As with another TLS polymerase, Pol IV, increasing concentrations of Pol II displace the Pol III core during DNA synthesis in a minimal reconstitution of primer extension. However, in contrast to Pol IV, Pol II is inefficient at disrupting rolling-circle synthesis by the fully reconstituted Pol III replisome. Together, these data suggest a β-mediated mechanism of exchange between Pol II and Pol III that occurs outside the replication fork.
大肠杆菌有三种与DNA损伤绕过有关的DNA聚合酶，这一过程称为跨损伤合成（TLS），可缓解复制停滞。尽管这些聚合酶专门针对不同的DNA损伤，但它们与复制机制的相互作用是否不同尚不清楚。在这三种聚合酶中，DNA聚合酶（Pol）II仍然是最神秘的。在此，我们报道了Pol II、复制性聚合酶Pol III核心复合物和二聚体持续性夹子β的稳定三元复合物。单分子实验表明，Pol II和Pol III与β的相互作用使得在DNA合成过程中能够快速交换。与另一种跨损伤合成聚合酶Pol IV一样，在引物延伸的最小重构中，DNA合成过程中Pol II浓度的增加会取代Pol III核心。然而，与Pol IV不同的是，Pol II在破坏完全重构的Pol III复制体的滚环合成方面效率较低。这些数据共同表明了一种由β介导的Pol II和Pol III之间的交换机制，该机制发生在复制叉之外。
